Method for preparing natural killer cells and use thereof

Abstract

The present disclosure relates to a method for rapidly culturing NK / CAR-NK cells, thereby obtaining NK / CAR-NK cells having very strong expansion and killing capacities.

Description

A method for preparing natural killer cells and its application

[0001] Cross-references to related applications

[0002] This application claims priority to International Application No. PCT / CN2025 / 074804, filed on January 24, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of biomedicine, specifically relating to a rapid preparation method for natural killer (NK) cells and their applications. Background Technology

[0004] Over the past 20 years, CAR-NK cells, as a novel type of immune cell, have been extensively studied. Allogeneic NK cells can exert therapeutic effects without causing acute graft-versus-host disease (GVHD). Compared with CAR-T cell therapy, the cytotoxicity and adverse events of CAR-NK cell therapy are easier to control (Romee R, et al. Sci Transl Med. 2016 Sep 21; 8(357):357ra123; Gang M, et al. Blood. 2020 Nov 12; 136(20):2308-2318.). Therefore, compared with CAR-T cells, the biological characteristics of NK cells make them a more promising universal immune cell therapy. Numerous clinical trials have validated the safety and efficacy of NK / CAR-NK cells as allogeneic cell therapy products in clinical use (Albinger N, et al. Blood Cancer J. 2022 Apr 13; 12(4):61; Shah N, et al. Clin Lymphoma Myeloma Leuk. 2014 Sep; 14Suppl(Suppl):S18-22; Marin D, et al. Nat Med. 2024 Mar; 30(3):772-784.).

[0005] Although CAR-NK cell products have demonstrated good safety and clinical efficacy in hematologic malignancies and solid tumors in clinical trials, the rapid elimination of allogeneic cells by the host immune system results in a short survival time for these cells in the body, leading to insufficient clinical efficacy. Therefore, improving the expansion and killing capacity of CAR-NK / NK cells in subjects has become an urgent problem to be solved in the development of CAR-NK cell products.

[0006] Overview

[0007] This disclosure provides a method for rapidly culturing NK cells, thereby obtaining NK cells with very strong proliferative and killing capabilities.

[0008] In one aspect, this disclosure provides a method for preparing natural killer (NK) cells, the method comprising co-culturing modified feeder cells with seed cells of NK cells, wherein the NK cells are cultured for no more than 10 days.

[0009] In some embodiments, the NK cells are cultured for 4 to 10 days; preferably, the NK cells are cultured for 6 to 9 days; more preferably, the NK cells are cultured for 6, 7, 8 or 9 days.

[0010] In some embodiments, the seed cells are selected from peripheral blood samples, peripheral blood mononuclear cell (PBMC) samples, umbilical cord blood samples, umbilical cord blood mononuclear cell (CBMC) samples, lymphocyte samples, leukocyte samples, apheresis products, leukocyte apheresis products, whole blood samples, erythrocyte sedimentation rate (ESR) brown-yellow layer samples, enriched or isolated primary NK cells, or NK cell lines; preferably, the seed cells are selected from apheresis products, leukocyte apheresis products, umbilical cord blood samples, umbilical cord blood mononuclear cell (CBMC) samples, peripheral blood samples, or peripheral blood mononuclear cell (PBMC) samples.

[0011] In some embodiments, the feeder cells are cell lines or primary cells.

[0012] In some embodiments, the feeder cells are K562, Jurkat, HuT-78, 721.221, SupT-1, or Nalm-6.

[0013] In some embodiments, the feeder cells are primary T cells.

[0014] In some embodiments, the modified feeder cells are irradiated to inactivate them.

[0015] In some embodiments, the feeder cells express at least one of the following: 4-1BB binding protein (such as 4-1BB ligand (4-1BBL) or 4-1BB antibody), membrane-bound interleukin-21 (mbIL-21), CD226 binding protein (such as CD226 antibody), NKG2A binding protein (such as NKG2A antibody), NKG2D binding protein (such as NKG2D antibody), IL-15 or a fusion protein of IL-15 receptor α (IL-15Rα), NKp30 binding protein (such as NKp30 antibody), IL-7, and IL-2.

[0016] In some embodiments, the feeder cells express 4-1BB ligand (4-1BBL) and membrane-bound interleukin-21 (mbIL-21).

[0017] In some embodiments, the feeder cells express: 4-1BBL and mbIL-21; CD226 antibody, 4-1BBL and mbIL-21; NKG2A antibody, 4-1BBL and mbIL-21; NKG2D antibody, 4-1BBL and mbIL-21; a fusion protein of IL-15 and IL-15Rα, 4-1BBL and mbIL-21; 4-1BB antibody and mbIL-21; NKp30 antibody, 4-1BBL and mbIL-21; IL-7; IL-2 and IL-7; 4-1BBL and IL-2; 4-1BBL; 4-1BBL and IL-7; IL-15; 4-1BBL and IL-15; or, IL-2 and IL-15.

[0018] In some embodiments, the seed cells are cultured in a mixture of the modified feeder cells at a ratio of 1:0.1 to 1:100.

[0019] In some embodiments, the seed cells are enriched or isolated primary NK cells.

[0020] In some embodiments, the seed cells and the modified feeder cells are cultured together at a ratio of 1:0.1 to 1:10; preferably, the ratio of the seed cells to the modified feeder cells is 1:0.5 to 1:4; more preferably, the ratio of the seed cells to the modified feeder cells is 1:0.5 to 1:2, such as 1:0.5 to 1:1, 1:0.5 to 1:1.5, 1:1 to 1:2, 1:1.5 to 1:2, or, for example, 1:1.

[0021] In some embodiments, the seed cells are peripheral blood mononuclear cell (PBMC) samples or apheresis products.

[0022] In some embodiments, the NK cells are prepared in a culture medium supplemented with cytokines; preferably, the cytokines are selected from one or more of IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, and IL-21.

[0023] In some embodiments, the method further includes introducing heterologous nucleic acids into NK cells or their seed cells.

[0024] In some embodiments, the heterologous nucleic acid encodes a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0025] In some implementations, the chimeric antigen receptor (CAR) specifically binds to the target antigen.

[0026] In some embodiments, the target antigen is selected from BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folic acid receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 fine adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, DLL3, CD70, CS-1, c-Met, glycolipid FF77, PD-L1 and PD-L2.

[0027] In some implementations, the heterologous nucleic acid is delivered via a lentiviral vector or a retroviral vector.

[0028] On the other hand, this disclosure provides natural killer (NK) cells prepared by the method for preparing natural killer (NK) cells described herein.

[0029] In some implementations, the NK cells have a higher CD25 positivity rate compared to NK cells prepared for more than 12 days using the same feeder cells.

[0030] In some implementations, the NK cells have a lower CD57 positivity rate compared to NK cells prepared for more than 16 days using the same feeder cells.

[0031] In some implementations, the NK cells have a lower CD16 positivity rate compared to NK cells prepared for more than 12 days using the same feeder cells.

[0032] In some implementations, the NK cells secrete higher levels of IFN-γ compared to NK cells cultured for more than 12 days using the same feeder cells.

[0033] In some implementations, the NK cells have a stronger killing ability compared to NK cells cultured for more than 12 days when co-cultured with the same feeder cells.

[0034] In some implementations, the NK cells exhibit a greater capacity for expansion after administration to a subject compared to NK cells cultured for more than 12 days using the same feeder cells.

[0035] In some implementations, the NK cells have stronger glycolytic capacity and / or glycolytic potential compared to NK cells cultured for more than 12 days in the same feeder cells.

[0036] In some implementations, at least a portion of the NK cells are further engineered to express IL-15.

[0037] In some embodiments, the IL-15 is secreted IL-15 or membrane-bound IL-15 (mbIL-15).

[0038] In another aspect, this disclosure provides NK cells suitable for administration to a subject in need, wherein (1) at least 40% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the NK cells express CD25; (2) at most 50% (e.g., at most 40%, at most 30%, at most 20%, at most 10%, or 0%) of the NK cells express CD57; and / or (3) at most 80% (e.g., at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, at most 10%, or 0%) of the NK cells express CD16.

[0039] In another aspect, this disclosure provides pharmaceutical compositions comprising NK cells as described herein or NK cells suitable for administration to a subject in need, and optionally pharmaceutically acceptable carriers and / or excipients.

[0040] In another aspect, this disclosure provides a method for treating a disease or condition, the method comprising administering to an individual in need an effective amount of the NK cells described herein, suitable for administration to a subject in need of the NK cells or the pharmaceutical composition described herein; preferably, the disease or condition is a tumor or an autoimmune disease.

[0041] In some implementations, the applied cells are autologous or allogeneic. Attached Figure Description

[0042] Figure 1A shows the fold expansion of CAR-NK cells harvested from Donor 570 at different time points when cultured with K562 feeder cells transduced with 4-1BBL and mbIL-21.

[0043] Figure 1B shows the fold expansion of CAR-NK cells harvested from Donor 571 at different time points when cultured with K562 feeder cells transduced with 4-1BBL and mbIL-21.

[0044] Figure 2A shows the tumor clearance rate of BCMA CAR-NK cells harvested from Donor 570 at different time points after three consecutive rounds of killing NCI-H929 cells.

[0045] Figure 2B shows the tumor clearance rate of BCMA CAR-NK cells harvested from Donor 570 at different time points after seven consecutive rounds of killing NCI-H929 cells.

[0046] Figure 2C shows the tumor clearance rate of BCMA CAR-NK cells harvested from Donor 571 at different time points after three consecutive rounds of killing NCI-H929 cells.

[0047] Figure 2D shows the tumor clearance rate of BCMA CAR-NK cells harvested from Donor 571 at different time points after seven consecutive rounds of killing NCI-H929 cells.

[0048] Figure 3A shows the cell expansion fold of BCMA CAR-NK cells harvested from Donor 570 at different time points after three consecutive rounds of killing NCI-H929 cells.

[0049] Figure 3B shows the fold expansion of BCMA CAR-NK cells harvested from Donor 570 at different time points after 72 hours of culture alone.

[0050] Figure 3C shows the cell expansion fold of BCMA CAR-NK cells harvested from Donor 571 at different time points after three consecutive rounds of killing NCI-H929 cells.

[0051] Figure 3D shows the fold expansion of BCMA CAR-NK cells harvested from Donor 571 at different time points after 72 hours of culture alone.

[0052] Figure 4A shows the amount of INF-γ secreted by BCMA CAR-NK / unNK cells harvested from Donor 570 at different time points 72 h after stimulation with and without tumor cells.

[0053] Figure 4B shows the amount of INF-γ secreted by BCMA CAR-NK / unNK cells harvested from Donor 571 at different time points 72 h after stimulation with and without tumor cells.

[0054] Figure 5A shows the basal metabolic activity of CAR-NK cells harvested from Donor 570 at different in vitro culture time points.

[0055] Figure 5B shows the basal metabolic activity of CAR-NK cells harvested from Donor 571 at different in vitro culture time points.

[0056] Figure 5C shows the glycolytic capacity of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points.

[0057] Figure 5D shows the glycolytic potential of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points.

[0058] Figure 6A shows the basal metabolic activity of CAR-NK cells harvested from Donor 570 at different in vitro culture time points after antigen stimulation.

[0059] Figure 6B shows the basal metabolic activity of CAR-NK cells harvested from Donor 571 at different in vitro culture time points after antigen stimulation.

[0060] Figure 6C shows the glycolytic capacity of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points after antigen stimulation.

[0061] Figure 6D shows the glycolytic potential of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points after antigen stimulation.

[0062] Figure 7A shows the expression level of CD16 in CAR-NK cells harvested at different in vitro culture time points.

[0063] Figure 7B shows the expression level of CD25 in CAR-NK cells harvested at different in vitro culture time points.

[0064] Figure 7C shows the expression level of CD57 in CAR-NK cells harvested at different in vitro culture time points.

[0065] Figure 8 shows the tumor clearance capacity of CD19 / BCMA CAR-NK cells harvested at different in vitro culture time points in a mouse model.

[0066] Figure 9 shows the amplification capacity of CD19 / BCMA CAR-NK cell products harvested at different in vitro culture time points in a mouse model.

[0067] Figure 10 shows the ability of BCMA CAR-NK cells harvested at different time points from genetically modified primary T cells as feeder cells to kill NCI-H929 cells in vitro.

[0068] Figure 11 shows the tumor clearance capacity of BCMA CAR-NK cells harvested at different in vitro culture time points in a mouse model.

[0069] Detailed Explanation

[0070] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise specified, implementation of certain embodiments or features of this disclosure may be carried out using conventional techniques of molecular biology, microbiology, recombinant DNA, etc., within the capabilities of those skilled in the art. Meanwhile, for a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0071] As used herein, the term "feeder cell" is also referred to as a helper cell for culture. In some cases, feeder cells refer to metabolically active cells that do not proliferate, thereby enabling the cell to produce various metabolites and contribute to the proliferation of target cells. Feeder cells may be inactivated by irradiation. Feeder cells can be cell lines or primary cells. Feeder cells can be tumor cells, such as K562, Jurkat, HuT-78, Nalm-6, etc. Feeder cells can be modified primary T cells, and the T cells used as feeder cells can be inactivated cells with inhibited division / proliferation or cells that have not been inactivated. Preferably, the T cells can be inactivated to ensure safety.

[0072] As used herein, the term "primary T cells" refers to T cells isolated directly from and / or stored (e.g., cryopreserved or cryopreserved) from a biological sample from a donor. The biological sample may be a sample obtained directly from a biological source or a processed sample, the processing of which may include separation, centrifugation, washing, storage, thawing, etc. Biological samples from which primary T cells can be obtained are well known to those skilled in the art and include, but are not limited to, peripheral blood samples, peripheral blood mononuclear cell (PBMC) samples, umbilical cord blood samples, umbilical cord blood mononuclear cell (CBMC) samples, lymphocyte samples, leukocyte samples, apheresis products, leukocyte apheresis products, whole blood samples, or erythrocyte sedimentation rate (ESR) amber samples. In the case of cell therapy (e.g., adoptive cell therapy), the sample includes samples from autologous or allogeneic sources.

[0073] As used herein, the term "seed cell" refers to a cell that can be expanded into NK cells through appropriate culture. Such cells are well known to those skilled in the art, and exemplary seed cells include, but are not limited to, peripheral blood samples, peripheral blood mononuclear cell (PBMC) samples, cord blood samples, cord blood mononuclear cell (CBMC) samples, lymphocyte samples, leukocyte samples, apheresis products, leukocyte apheresis products, whole blood samples, erythrocyte sedimentation rate (ESR) amber layer samples, enriched or isolated primary NK cells, or NK cell lines.

[0074] As used herein, the term "culture medium" has the meaning commonly understood by those skilled in the art as referring to a solution containing nutrients necessary for cell growth. Typically, such solutions provide at least the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for cell growth and / or survival.

[0075] As used herein, the expression "positive for a specific marker" means the detectable presence of a specific marker (typically a surface marker) on or within a cell. When referring to a surface marker, the term means the presence of surface expression as detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting the antibody.

[0076] As used herein, the expression "negative for a specific marker" means that the specific marker (typically a surface marker) is not substantially detectably present on or within the cell. When referring to a surface marker, the term means the absence of surface expression, as detected by flow cytometry, for example by staining with an antibody that specifically binds to the marker and detecting the antibody.

[0077] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. This term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, scFv), nanobodies, fusion proteins including antibodies, and any other modified conformations of immunoglobulin molecules including antigen recognition sites. The VH and VL regions of an antibody can be further subdivided into highly denatured regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residue in the variable region of the antibody responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system.

[0078] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding moiety." Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, nanobodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0079] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0080] As used in this article, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells such as T cells and NK cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CARs can contain an extracellular antigen-binding domain specific to one or more antigens (such as B cell antigens), a transmembrane domain, and an intracellular signaling domain for T cells and / or other receptors. "CAR-NK cells" refers to NK cells that express CARs.

[0081] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). Vectors may contain various elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. "Retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof primarily derived from retroviruses. "Lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof (including LTRs) primarily derived from lentiviruses.

[0082] As used herein, the terms “subject” and “patient” are used interchangeably. A subject can be a mammal, such as a non-primate or a primate (e.g., a human). A subject can be a human. A subject can be a mammal diagnosed with a disease or condition, such as a human. A subject can be a mammal at risk of developing a disease or condition, such as a human.

[0083] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, is well known in the art, and includes, but is not limited to: pH adjusters, surfactants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Agents for delaying absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. The carrier and / or excipient may be sterile water, physiological saline, glucose, human serum albumin (HSA), dimethyl sulfoxide (DMSO), dextran, or analogues thereof (e.g., dextran 40).

[0084] As used herein, the term "treatment" refers to a method performed to achieve a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes (but is not limited to) relief of symptoms, reduction of the extent of disease, stabilization (i.e., cessation of disease progression) of the disease state, delay or slowing of disease progression, improvement or relief of the disease state, and relief of symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0085] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, a therapeutically effective amount is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the state of the patient's immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0086] As used in this article, the term “donor” can be any individual, such as a living organism capable of triggering an immune response, such as a mammal, such as a human.

[0087] "Cultivation days" refers to the total time required to culture natural killer (NK) cells from seed cells until the desired outcome (e.g., cell expansion, functional maturation) is achieved. The time when the seed cells of NK cells are added to the culture medium is defined as Day 0, regardless of the specific time of day. Depending on experimental requirements, feeder cells can be co-cultured with the seed cells of NK cells at an appropriate time (e.g., Day 0). During cultivation, feeder cells can be removed at appropriate points without affecting subsequent NK cell cultivation. Furthermore, depending on the experimental objective, heterologous nucleic acids of interest (e.g., CARs) can be delivered into the cells via vectors during cultivation. When NK cells are harvested on day n, the cultivation days are n days (n-0), regardless of the specific time of day. For example, if seed cells of NK cells are added to the culture medium at 8:00 AM on Day 0 and NK cells are harvested at 5:00 PM on Day 7, the cultivation cycle is counted as 7 days. Similarly, if the seed cells of NK cells are added to the culture medium at 5 pm on Day 0 and the NK cells are harvested at 8 am on Day 7, the culture cycle is also counted as 7 days.

[0088] This disclosure provides a method for rapidly culturing NK / CAR-NK cells, thereby obtaining NK / CAR-NK cells with very strong proliferative and cytotoxic capabilities. Due to the host immune system's clearance (HvG), the expansion window of allogeneic cell therapy products is typically only about 10 days (7-14 days). The method provided in this disclosure can enhance the in vivo expansion capacity of allogeneic NK cell products, and is expected to achieve higher maximum plasma drug concentrations (Cmax) within the limited expansion window, thereby increasing drug exposure and improving the clinical therapeutic effect of allogeneic cell therapy products.

[0089] Feeder cells

[0090] According to one aspect, this disclosure provides a method for preparing NK cells, wherein the method includes co-culturing feeder cells with seed cells of NK cells.

[0091] In some embodiments, the feeder cells are cell lines or primary cells.

[0092] In some embodiments, the cell line is a tumor cell. In some embodiments, the feeder cells are K562, Jurkat, HuT-78, 721.221, SupT-1, Nalm-6, HL-60, AML3, Daudi, JAWSII, RPMI8866, HFWT, EBV_LCL, or NK-92. In some embodiments, the feeder cells are K562, Jurkat, HuT-78, 721.221, SupT-1, or Nalm-6. In some embodiments, the feeder cells are K562.

[0093] In some embodiments, the feeder cells are primary T cells. In some embodiments, the primary T cells are CD3+ T cells enriched or isolated from a donor's biological sample. For example, they can be obtained through positive selection based on a CD3-binding reagent (e.g., magnetic beads coated with CD3 antibodies). In some embodiments, the primary T cells contain CD4+ T cells and / or CD8+ T cells. In some embodiments, the biological sample is or includes a peripheral blood sample, a peripheral blood mononuclear cell (PBMC) sample, a cord blood sample, a cord blood mononuclear cell (CBMC) sample, a lymphocyte sample, a leukocyte sample, apheresis product, leukocyte apheresis product, a whole blood sample, or erythrocyte sedimentation rate (ESR) amber layer sample. In some embodiments, the biological sample is an apheresis product or a leukocyte apheresis product. The biological sample can be a sample obtained directly from a biological source or a processed sample, the processing of which can include separation, centrifugation, washing, storage (e.g., cryopreservation or cryopreservation), thawing, etc. In some embodiments, the donor is a human.

[0094] In some embodiments, the modified feeder cells express 4-1BB ligand (4-1BBL) and membrane-bound interleukin-21 (mbIL-21). In some embodiments, the modified feeder cells express both 4-1BBL and mbIL-21. mbIL-21 refers to IL-21 that can bind to the cell membrane. Such protein forms of IL-21 are known to those skilled in the art; for example, mbIL-21 can be a fusion protein formed by the binding of IL-21 to a transmembrane protein. The transmembrane protein can be CD8α, specifically the transmembrane domain of CD8α. In some exemplary embodiments, the amino acid sequences of 4-1BBL and mbIL-21 can be found in PCT patent application PCT / CN2024 / 072753, the disclosure of which is incorporated herein by reference.

[0095] In some embodiments, the modified feeder cells are irradiated to inactivate them, for example, by irradiating them with X-rays or gamma rays to render them incapable of proliferation.

[0096] Preparation of feeder cells

[0097] According to one aspect, this disclosure relates to a method for preparing feeder cells for culturing natural killer (NK) cells, wherein a foreign nucleic acid molecule is introduced into the feeder cells (e.g., K562 or primary T cells) to obtain modified feeder cells (e.g., modified K562 or modified primary T cells), which are the feeder cells. The foreign nucleic acid molecule comprises a nucleotide sequence encoding at least one foreign protein selected from: 4-1BB binding protein (e.g., 4-1BB ligand (4-1BBL) or 4-1BB antibody), membrane-bound interleukin-21 (mbIL-21), CD226 binding protein (e.g., CD226 antibody), NKG2A binding protein (e.g., NKG2A antibody), NKG2D binding protein (e.g., NKG2D antibody), IL-15 or a fusion protein thereof with IL-15 receptor α (IL-15Rα), NKp30 binding protein (e.g., NKp30 antibody), IL-7, IL-2, or any combination thereof. The amino acid sequence of the exogenous protein can be found in PCT patent application PCT / CN2024 / 072753, the contents of which are incorporated herein by reference.

[0098] In some embodiments, the nucleotide sequence encoding the foreign protein may be operatively linked to an expression regulatory element (e.g., a promoter).

[0099] In some embodiments, the exogenous nucleic acid molecule comprises a nucleotide sequence encoding a 4-1BB ligand (4-1BBL) and a nucleotide sequence encoding a membrane-bound interleukin-21 (mbIL-21).

[0100] In some embodiments, the exogenous nucleic acid molecule further comprises a nucleotide sequence encoding at least one exogenous protein selected from the following: CD226-binding protein (such as CD226 antibody), NKG2A-binding protein (such as NKG2A antibody), NKG2D-binding protein (such as NKG2D antibody), IL-15, or a fusion protein thereof with IL-15 receptor α (IL-15Rα).

[0101] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding 4-1BBL and mbIL-21.

[0102] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding the CD226 antibody, 4-1BBL, and mbIL-21.

[0103] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding NKG2A antibody, 4-1BBL, and mbIL-21.

[0104] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding NKG2D antibody, 4-1BBL, and mbIL-21.

[0105] In some embodiments, the exogenous nucleic acid molecule comprises nucleotide sequences encoding a fusion protein of IL-15 and IL-15Rα, 4-1BBL, and mbIL-21.

[0106] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding 4-1BB antibody and mbIL-21.

[0107] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding NKp30 antibody, 4-1BBL, and mbIL-21.

[0108] In some embodiments, the exogenous nucleic acid molecule contains a nucleotide sequence encoding IL-7.

[0109] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding IL-2 and IL-7.

[0110] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding 4-1BBL and IL-2.

[0111] In some embodiments, the exogenous nucleic acid molecule contains a nucleotide sequence encoding 4-1BBL.

[0112] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding 4-1BBL and IL-7.

[0113] In some embodiments, the exogenous nucleic acid molecule contains a nucleotide sequence encoding IL-15.

[0114] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding 4-1BBL and IL-15.

[0115] In some embodiments, the exogenous nucleic acid molecule contains nucleotide sequences encoding IL-2 and IL-15.

[0116] In some embodiments, the feeder cells are K562. In some embodiments, the step of introducing the exogenous nucleic acid molecule includes introducing the exogenous nucleic acid molecule into the K562 cells via a viral vector (e.g., a lentivirus or retrovirus vector). In some embodiments, the introduction is a viral vector-mediated transfection.

[0117] In some embodiments, the method further includes irradiating the modified K562 to inactivate the cells. This can be done, for example, by irradiating it with X-rays or gamma rays to disable its proliferative capacity. In some embodiments, the irradiation dose is 100–300 Gy.

[0118] In some implementations, the nucleic acid molecules are cultured for 3 to 25 days, for example 3 to 20 days, for example 5 to 25 days, for example 5 to 20 days, for example 5 to 15 days.

[0119] In some embodiments, the method involves culturing K562 cells in a culture medium. The culture medium can be any medium suitable for mammalian cells. Many such media are commercially available; exemplary media include IMDM, RPMI 1640, DMEM, MEM, AIM-V, TexMACS, X-VIVO 15, PRIME-XV T Cell CDM, etc. The culture medium may be supplemented with one or more of the following: growth factors, cytokines, hormones, antibiotics, vitamins, etc. The culture medium may be serum-free or serum-containing.

[0120] In some embodiments, the method further includes using flow cytometry to detect the expression of the aforementioned exogenous protein in the modified K562.

[0121] In some embodiments, the feeder cells are primary T cells. In some embodiments, the step of isolating primary T cells from a donor biological sample includes enriching or isolating CD3+ T cells from the biological sample to obtain primary T cells. For example, this can be achieved through positive selection based on a CD3-binding reagent (e.g., magnetic beads coated with CD3 antibodies). In some embodiments, the step of introducing exogenous nucleic acid molecules includes introducing the exogenous nucleic acid molecules into the primary T cells via a viral vector (e.g., a lentivirus or retroviral vector). In some embodiments, the introduction is a viral vector-mediated transfection.

[0122] In some embodiments, the method further includes irradiating the modified primary T cells to inactivate them. This can be done, for example, by irradiating them with X-rays or gamma rays to render them incapable of proliferation. In some embodiments, the irradiation dose is 100–300 Gy.

[0123] In some embodiments, the obtained primary T cells are further pretreated before the introduction of exogenous nucleic acid molecules, the pretreatment including activation and / or proliferation. In some embodiments, the pretreatment includes contacting the primary T cells with an activating agent, for example, culturing the primary T cells in a culture medium supplemented with the activating agent. In some embodiments, the activating agent includes anti-CD3 antibodies and anti-CD28 antibodies.

[0124] In some embodiments, the method includes: contacting and incubating primary T cells isolated from a donor's biological sample with an activating agent (e.g., anti-CD3 antibody and anti-CD28 antibody) for 1–2 days; introducing the exogenous nucleic acid molecule into the primary T cells incubated with the activating agent and culturing for at least 5 days to obtain modified primary T cells.

[0125] In some implementations, the nucleic acid molecules are cultured for 3 to 25 days, for example 3 to 20 days, for example 5 to 25 days, for example 5 to 20 days, for example 5 to 15 days.

[0126] In some embodiments, the method involves culturing primary T cells in a culture medium. The culture medium can be any medium suitable for mammalian cells. Many such media are commercially available; exemplary media include RPMI 1640, DMEM, MEM, AIM-V, TexMACS, X-VIVO 15, PRIME-XV T Cell CDM, etc. The culture medium may be supplemented with one or more of the following: growth factors, cytokines, hormones, antibiotics, vitamins, etc. The culture medium may be serum-free or serum-containing.

[0127] In some embodiments, the method further includes using flow cytometry to detect the expression of the aforementioned exogenous protein in modified primary T cells.

[0128] Culture or expand NK cells

[0129] According to one aspect, this disclosure relates to a method for preparing NK cells, wherein the method includes co-culturing modified feeder cells with seed cells of NK cells, wherein the NK cells are cultured for no more than 10 days.

[0130] The time when the seed cells of NK cells are added to the culture medium is defined as day 0. When the NK cells are harvested on day 10, the culture period is 10 days (10-0), without considering the specific time point.

[0131] In some embodiments, the NK cells are cultured for 4 to 10 days. In some embodiments, the NK cells are cultured for 6 to 9 days. In some embodiments, the NK cells are cultured for 6, 7, 8, or 9 days.

[0132] Traditional NK cell production processes typically take 14 to 21 days, while the method disclosed herein shortens the production cycle to 6 to 10 days. This not only significantly improves the utilization rate of GMP facilities and equipment but also reduces labor costs, thereby greatly enhancing the future competitiveness of the product in terms of production costs.

[0133] Feeder cells are helper cells used in culture that help expand target cells.

[0134] In some embodiments, the feeder cells are the modified K562 described in this disclosure.

[0135] In some embodiments, the feeder cells are the modified primary T cells described in this disclosure. In some embodiments, the modified primary T cells and the NK cells are derived from the same donor. In some embodiments, the modified primary T cells and the NK cells are derived from different donors.

[0136] Seed cells are cells that can be expanded into NK cells through appropriate culture. Such cells are well known to those skilled in the art, and exemplary seed cells include peripheral blood samples, peripheral blood mononuclear cell (PBMC) samples, cord blood samples, cord blood mononuclear cell (CBMC) samples, lymphocyte samples, leukocyte samples, apheresis products, leukocyte apheresis products, whole blood samples, erythrocyte sedimentation rate (ESR) amber layer samples, enriched or isolated primary NK cells, or NK cell lines.

[0137] In some embodiments, the seed cells are selected from apheresis products, leukocyte apheresis products, cord blood samples, cord blood mononuclear cell (CBMC) samples, peripheral blood samples, or peripheral blood mononuclear cell (PBMC) samples.

[0138] In some embodiments, the seed cells and the feeder cells are cultured in a ratio of 1:0.1 to 1:100 (e.g., 1:0.1 to 1:80, 1:0.1 to 1:50, 1:0.1 to 1:40, 1:0.1 to 1:20, 1:0.1 to 1:10, 1:0.2 to 1:80, 1:0.2 to 1:50, 1:0.2 to 1:40, 1:0.2 to 1:20, 1:0.2 to 1:10, 1:0.5 to 1:80, 1:0.5 to 1:50, 1:0.5 to 1:40, 1:0.5 to 1:20, 1:0.5 to 1:10). In some embodiments, the seed cells are enriched or isolated primary NK cells.

[0139] In some embodiments, the seed cells and feeder cells are cultured together at a ratio of 1:0.1 to 1:10 (e.g., 1:0.1 to 1:8, 1:0.1 to 1:5, 1:0.1 to 1:4, 1:0.2 to 1:10, 1:0.2 to 1:8, 1:0.2 to 1:5, 1:0.2 to 1:4, 1:0.5 to 1:10, 1:0.5 to 1:8, 1:0.5 to 1:5, 1:0.5 to 1:4). In some embodiments, the seed cells and feeder cells are cultured together at a ratio of 1:0.5 to 1:4. In some embodiments, the ratio (i.e., the number ratio) of the seed cells to the feeder cells is 1:0.5 to 1:2, for example, 1:0.5 to 1:1, 1:0.5 to 1:1.5, 1:1 to 1:2, 1:1.5 to 1:2, for example, 1:1. In some embodiments, the seed cells are peripheral blood mononuclear cell (PBMC) samples or apheresis products.

[0140] In some embodiments, the culture is performed in a medium supplemented with cytokines. The medium can be any medium suitable for mammalian cells. Many such media are commercially available; exemplary media include KBM581, RPMI1640, X-VIVO15, AIM-V, NK MACS, CTS NK-Xpander, PRIME-XV NK Cell CDM, etc. The medium may be supplemented with one or more of the following: growth factors, cytokines, hormones, antibiotics, vitamins, etc. The medium may be serum-free or serum-containing.

[0141] In some embodiments, the cytokine is selected from one or more of IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, and IL-21. In some exemplary embodiments, the cytokine includes IL-2 and / or IL-15. In some exemplary embodiments, the concentration of the cytokine is 10 U / mL to 2000 U / mL, for example, 100 U / mL to 2000 U / mL, 100 U / mL to 1000 U / mL, or 100 U / mL to 500 U / mL. In some exemplary embodiments, the concentration of the cytokine is 1 ng / mL to 1000 ng / mL, for example 1 ng / mL to 800 ng / mL, 1 ng / mL to 500 ng / mL, 1 ng / mL to 200 ng / mL, 1 ng / mL to 100 ng / mL, 10 ng / mL to 1000 ng / mL, 10 ng / mL to 800 ng / mL, 10 ng / mL to 500 ng / mL, 10 ng / mL to 200 ng / mL, or 10 ng / mL to 100 ng / mL. In some exemplary embodiments, the concentration of the cytokine is 1 IU / mL to 2000 IU / mL, for example, 1 IU / mL to 1500 IU / mL, 1 IU / mL to 1000 IU / mL, 1 IU / mL to 500 IU / mL, 10 IU / mL to 1500 IU / mL, 10 IU / mL to 1000 IU / mL, 10 IU / mL to 500 IU / mL, 100 IU / mL to 1500 IU / mL, 100 IU / mL to 1000 IU / mL, 100 IU / mL to 500 IU / mL, 500 IU / mL to 1000 IU / mL, 500 IU / mL to 1500 IU / mL. It is known in the art that units such as U / mL, IU / mL, and ng / mL can all be used to describe the concentration of cytokines. Preferably, the unit U / mL or IU / mL is used when describing the concentration of IL-2. Preferably, the unit ng / mL is used when describing the concentration of cytokines such as IL-15, IL-18, and IL-21. In some exemplary embodiments, the cytokines include IL-2 at 500 IU / mL to 1500 IU / mL and / or IL-15 at 10 ng / mL to 100 ng / mL. In some exemplary embodiments, the cytokines include IL-2 at 1000 IU / mL.

[0142] In some embodiments, culturing for 6 days yields at least a 10-fold amplification, such as at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 50-fold, or at least 60-fold amplification. In some embodiments, culturing for 6 days yields at least a 25-fold amplification.

[0143] In some embodiments, culturing for 7 days yields at least 50-fold amplification, such as at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 110-fold, at least 120-fold, at least 150-fold, at least 200-fold, at least 250-fold, or at least 300-fold amplification. In some embodiments, culturing for 7 days yields at least 120-fold amplification.

[0144] In some embodiments, culturing for 8 days yields at least 100-fold amplification, such as at least 120-fold, at least 150-fold, at least 180-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, or at least 500-fold amplification. In some embodiments, culturing for 8 days yields at least 200-fold amplification.

[0145] In some embodiments, culturing for 9 days yields at least 300-fold amplification, such as at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or at least 1500-fold. In some embodiments, culturing for 9 days yields at least 650-fold amplification.

[0146] In some embodiments, culturing for 10 days yields at least 800-fold amplification, such as at least 850-fold, at least 900-fold, at least 950-fold, at least 1000-fold, at least 1100-fold, at least 1200-fold, at least 1300-fold, at least 1400-fold, at least 1500-fold, at least 2000-fold, or at least 3000-fold amplification. In some embodiments, culturing for 10 days yields at least 1300-fold amplification.

[0147] In some embodiments, the NK cells obtained through the culture highly express CD25. In some embodiments, the NK cells obtained through the culture have a higher CD25 positivity rate compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days. In some embodiments, the NK cells obtained through the culture express higher levels of CD25 compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days. In some embodiments, at least 40% (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the NK cells obtained through the culture express CD25.

[0148] In some embodiments, the NK cells obtained through the culture show low expression of CD57. In some embodiments, the NK cells obtained through the culture show a lower CD57 positivity rate compared to NK cells prepared by co-culturing with the same feeder cells for more than 16 days. In some embodiments, the NK cells obtained through the culture show lower CD57 expression levels compared to NK cells prepared by co-culturing with the same feeder cells for more than 16 days. In some embodiments, up to 50% (e.g., up to 45%, up to 40%, up to 30%, up to 20%, up to 10%, or 0%) of the NK cells obtained through the culture express CD57.

[0149] In some embodiments, the NK cells obtained through the culture show low expression of CD16. In some embodiments, the NK cells obtained through the culture show a lower CD16 positivity rate compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days. In some embodiments, the NK cells obtained through the culture show lower CD16 expression levels compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days. In some embodiments, up to 80% (e.g., up to 75%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, or 0%) of the NK cells obtained through the culture express CD16.

[0150] In some embodiments, the NK cells obtained through the culture have higher IFN-γ secretion levels. In some embodiments, the NK cells obtained through the culture have higher IFN-γ secretion levels compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days.

[0151] In some embodiments, the NK cells obtained through the culture exhibit strong cytotoxicity. In some embodiments, the NK cells obtained through the culture have stronger cytotoxicity compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days.

[0152] In some embodiments, the NK cells obtained through the culture exhibit a strong proliferative capacity after being administered to a subject. In some embodiments, the NK cells obtained through the culture exhibit a stronger proliferative capacity after being administered to a subject compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days.

[0153] In some embodiments, the NK cells obtained through the culture exhibit strong glycolytic capacity and / or glycolytic potential. In some embodiments, the NK cells obtained through the culture have stronger glycolytic capacity and / or glycolytic potential compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days.

[0154] According to one aspect, this disclosure also relates to NK cells prepared by the methods described above. In some embodiments, the NK cells obtained by the methods for culturing NK cells described in this disclosure contain other impurity cells in proportions of at least <2%, such as <2%, <1.5%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.05%, or <0.01%.

[0155] Preparation of CAR-NK cells

[0156] According to one aspect, this disclosure relates to the preparation of NK cells expressing chimeric antigen receptors (CARs), including a cell culture step and a step of introducing a foreign nucleic acid molecule encoding the CAR, wherein the cell culture involves a method for culturing NK cells as described in this disclosure.

[0157] In some embodiments, a nucleic acid molecule encoding CAR is introduced into the seed cells of NK cells, which are then cultured using the NK cell culture method described in this disclosure.

[0158] In some embodiments, NK cells are obtained by culturing NK cells using the method for culturing NK cells described in this disclosure, and then a nucleic acid molecule encoding CAR is introduced into the NK cells.

[0159] In some embodiments, the nucleic acid molecule encoding the CAR is introduced into cells via a viral vector (e.g., a lentiviral or retroviral vector). In some embodiments, the introduction is a viral vector-mediated transfection.

[0160] In some implementations, the nucleic acid molecule encoding the CAR is introduced into the cell via electroporation.

[0161] In some implementations, the CAR includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

[0162] An extracellular antigen-binding domain endows the CAR with the ability to recognize a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigen is an autoimmune disease antigen. In some embodiments, the extracellular antigen-binding domain comprises an antibody or an antigen-binding fragment thereof that specifically binds to the target antigen. In some embodiments, the extracellular antigen-binding domain includes, but is not limited to, Fab fragments, Fab' fragments, F(ab)'2 fragments, Fv, disulfide-stabilized Fv (“dsFv”), scFv, or nanobodies. In some embodiments, the target antigen is selected from BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folic acid receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 fine adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, DLL3, CD70, CS-1, c-Met, glycolipid FF77, PD-L1 and PD-L2.

[0163] Numerous CARs targeting different tumor antigens have been widely disclosed in the field of cell therapy, such as CD19 CARs and BCMA CARs. The extracellular antigen-binding domain of a CD19 CAR can be a CD19-binding fragment or contain a CD19-binding fragment (such as those disclosed in various patents like FMC63, SJ25C1, or WO 2022 / 012683). BCMA CARs have also been well described, with related patents including but not limited to WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760, and WO 2018 / 028647.

[0164] A transmembrane domain can be any protein structure that is thermodynamically stable within the cell membrane (particularly the eukaryotic cell membrane). In some embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: the α, β, or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD-1. In some embodiments, the transmembrane domain is a transmembrane region of CD8α.

[0165] Intracellular signal transduction domains are involved in transducing effective antigen-receptor binding signals into NK cells, activating at least one normal effector function of CAR-expressing NK cells, or enhancing the secretion of at least one cytokine from CAR-expressing NK cells. In some embodiments, the intracellular signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory signal transduction domain. In some embodiments, the primary signal transduction domain comprises an immune receptor tyrosine activation motif (ITAM). In some embodiments, the primary signal transduction domain comprises a signal transduction domain selected from the following proteins: CD3ζ, FcRγ, FcRβ, DAP12, DAP10, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, and any combination thereof. In some embodiments, the primary signal transduction domain is selected from CD3ζ. In some embodiments, the co-stimulatory signal transduction domain comprises a signal transduction domain selected from the following proteins: DAP12, DAP10, CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand, and any combination thereof. In some embodiments, the co-stimulatory signal transduction domain is selected from CD137. In some embodiments, the intracellular signal transduction domain comprises or consists of a CD137 co-stimulatory signal transduction domain and a CD3ζ primary signal transduction domain. In some embodiments, the intracellular signal transduction domain further includes a C-terminal DAP12 intracellular signal transduction domain. In some embodiments, the intracellular signal transduction domain comprises or consists of a CD13 co-stimulatory signal transduction domain, a CD3ζ primary signal transduction domain, and a DAP12 intracellular signal transduction domain.

[0166] In some embodiments, the intracellular signaling domains, from N-terminus to C-terminus, are sequentially: a CD137 signaling domain and a CD3ζ primary signaling domain. In some embodiments, the intracellular signaling domains, from N-terminus to C-terminus, are sequentially: a CD137 signaling domain, a CD3ζ primary signaling domain, and a DAP12 signaling domain. In some embodiments, the CAR further includes a spacer domain located between the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, the spacer domain is selected from hinge domains and / or the CH2 and CH3 regions of immunoglobulins (e.g., IgG1 or IgG4). The hinge domain can be an amino acid segment typically found between two domains of a protein, which allows the protein to be flexible and allows movement of one or both domains relative to each other. Therefore, the hinge domain can be any amino acid sequence, as long as it provides this flexibility of the extracellular antigen-binding domain and its mobility relative to the transmembrane domain. In some embodiments, the hinge domain comprises the hinge region of CD8α, CD28, or IgG4Fc. In some embodiments, the hinge structure domain includes the hinge region of CD8α.

[0167] In some embodiments, the CAR further includes a signal peptide at its N-terminus. Typically, a signal peptide is a polypeptide sequence to which a linked sequence is targeted to a desired site in the cell. In some embodiments, the signal peptide can target the linked CAR to the cellular secretory pathway and allow the CAR to further integrate and anchor into a lipid bilayer. Signal peptides that can be used with CARs are known to those skilled in the art. In some embodiments, the signal peptide comprises a CD8α signal peptide, a GM-CSF receptor α signal peptide, or a heavy chain signal peptide (e.g., the heavy chain signal peptide of IgG1).

[0168] In some embodiments, the CAR, from N-terminus to C-terminus, comprises: a signal peptide, an extracellular antigen-binding domain, a CD8α hinge region, a CD8α transmembrane region, a CD137 signal transduction domain, and a CD3ζ primary signal transduction domain.

[0169] In some embodiments, the CAR, from N-terminus to C-terminus, comprises: a signal peptide, an extracellular antigen-binding domain, a CD8α hinge region, a CD8α transmembrane region, a CD137 signal transduction domain, a CD3ζ primary signal transduction domain, and a DAP12 signal transduction domain.

[0170] According to one aspect, this disclosure also relates to NK cells expressing a chimeric antigen receptor (CAR), prepared by the methods described above. The CAR-expressing NK cells may be further armored. In some embodiments, the CAR-NK cells are further armored with an IL-15 peptide or a variant thereof. In some embodiments, the IL-15 is secreted IL-15 or membrane-bound IL-15 (mbIL-15). Non-limiting information regarding membrane-bound IL-15 can be found in PCT patent applications PCT / US2019 / 050679 and PCT / CN2024 / 124484, which are incorporated herein by reference in their entirety. The IL-15 or a variant thereof may be linked to the CAR via P2A.

[0171] According to one aspect, this disclosure also relates to cell compositions comprising NK cells expressing CAR as described above. In some embodiments, the cell composition may further comprise NK cells that do not express and / or have not successfully expressed the CAR. In some cases, not all immune cells are modified to express the target CAR; however, immune cells that do not express CAR still possess certain biological activity. Therefore, the cell compositions described in this disclosure may contain NK cells expressing and not expressing the target CAR, and the cell compositions can still meet the needs of clinical applications.

[0172] NK cell products

[0173] According to one aspect, this disclosure relates to NK cells suitable for administration to a subject in need, wherein (1) at least 40% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of the NK cells express CD25; (2) at most 50% (e.g., at most 40%, at most 30%, at most 20%, at most 10%, or 0%) of the NK cells express CD57; and / or (3) at most 80% (e.g., at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, at most 10%, or 0%) of the NK cells express CD16. The NK cells suitable for administration to a subject in need refer to NK cells that have been isolated from a culture medium and can be administered to a subject as a final product or in combination with an optional pharmaceutically acceptable carrier and / or excipient to form a pharmaceutical composition for administration to a subject.

[0174] Therapeutic applications

[0175] According to one aspect, this disclosure relates to pharmaceutical compositions comprising NK cells obtained by the methods for culturing NK cells described in this disclosure, CAR-NK cells obtained by the methods for preparing CAR-expressing NK cells described in this disclosure, NK cells suitable for administration to a subject in need, or cell compositions described in this disclosure; and optionally pharmaceutically acceptable carriers and / or excipients. In some embodiments, the pharmaceutical composition may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0176] According to one aspect, this disclosure relates to a method for treating a disease or condition (e.g., a tumor, an autoimmune disease), the method comprising administering to an individual in need an effective amount of NK cells obtained by the method of culturing NK cells as described in this disclosure, or CAR-NK cells obtained by the method of preparing CAR-expressing NK cells as described in this disclosure, NK cells suitable for administration to a subject in need, or a cell composition as described in this disclosure, or a pharmaceutical composition as described in this disclosure.

[0177] In some embodiments, the proportion of other impurity cells in NK cells obtained by the method of culturing NK cells described in this disclosure, or in CAR-NK cells obtained by the method of preparing CAR-expressing NK cells described in this disclosure, is at least <2%, for example <2%, <1.5%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.05%, or <0.01%.

[0178] In some implementations, the individual is a person.

[0179] In some implementations, the applied cells are autologous, meaning that the individual is the same as the donor of the applied cells.

[0180] In some implementations, the applied cells are allogeneic, meaning that the individual is not the same as the donor of the applied cells.

[0181] In some embodiments, the method includes the following steps: (1) providing seed cells of NK cells from a donor; (2) culturing the seed cells using the method for culturing NK cells described in this disclosure; (3) introducing a nucleic acid molecule encoding a CAR into the NK cells or the seed cells during the culture process; and (4) administering the cultured cell product (optionally formulated as a pharmaceutical composition) to an individual in need of treatment. In some embodiments, step (3) further includes introducing a gene encoding another protein, such as a cytokine, like IL-15 or a variant thereof, into the NK cells.

[0182] In some embodiments, the method includes the following steps: (1) providing seed cells of NK cells from a donor; (2) introducing a nucleic acid molecule encoding a CAR into the cells of step (1); (3) culturing the cell product of step (2) using the method for culturing NK cells described in this disclosure; and (4) administering the cell product of step (3) (optionally configured as a pharmaceutical composition) to an individual in need of treatment. In some embodiments, step (2) further includes introducing a gene encoding another protein, such as a cytokine, like IL-15 or a variant thereof, into the NK cells.

[0183] In some embodiments, the method includes the following steps: (1) providing seed cells from NK cells of a donor; (2) culturing the seed cells using the NK cell culture method described in this disclosure; (3) introducing a nucleic acid molecule encoding a CAR into the cell product of step (2); and (4) administering the cell product of step (3) (optionally configured as a pharmaceutical composition) to an individual in need of treatment. In some embodiments, step (3) further includes introducing a gene encoding another protein, such as a cytokine, like IL-15 or a variant thereof, into the NK cells.

[0184] The NK cells, CAR-NK cells, NK cell compositions suitable for administration to subjects in need, and pharmaceutical compositions described herein can be formulated into any dosage form known in the medical field, with preferred dosage forms depending on the intended route of administration and therapeutic use. In some embodiments, injectable formulations (e.g., solutions for injection) are preferred.

[0185] The NK cells, CAR-NK cells, NK cell compositions, and pharmaceutical compositions described herein can be administered by any suitable method known in medicine, such as intravenous, intraperitoneal, intratracheal, intrathecal, intratumoral, intramuscular, intratumoral, intramuscular, endoscopic, intralesional, percutaneous, subcutaneous, local, or by direct injection or infusion. In some embodiments, intravenous administration is preferred.

[0186] According to one aspect, this disclosure also relates to the use of NK cells obtained by the methods for culturing NK cells described herein, or CAR-NK cells obtained by the methods for preparing CAR-expressing NK cells described herein, NK cells suitable for administration to subjects in need, or cell compositions or pharmaceutical compositions described herein, in the preparation of a medicament for treating a disease or condition. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is an autoimmune disease. Example

[0187] The embodiments of this disclosure will now be described in detail with reference to examples. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by this disclosure. Various modifications and variations can be made to the details based on all published teachings, and all such modifications and variations are within the scope of protection of this disclosure.

[0188] Example 1: Preparation of Feeder Cells

[0189] The feeder cells in this invention can be genetically modified primary T cells, K562 cells, or other tumor cell lines (e.g., Jurkat, HuT-78, Nalm-6, etc.). Genetically modified primary T cells and genetically modified K562 cells are used as examples below.

[0190] 1.1 Preparation process of genetically modified K562 feeder cells

[0191] Construction of K562 cells stably overexpressing 4-1BBL and mbIL-21: K562 cells (purchased from ATCC) were resuscitated and cultured for 1-3 days. The cells were then transduced using a lentiviral vector containing the target genes for 4-1BBL and mbIL-21. Twenty-four hours after transduction, the cells were washed once with PBS, resuspended in culture medium, and seeded into 6-well plates or T-25 flasks. After 72 hours, a small number of cells were taken and incubated with the flow cytometry antibodies Anti-4-1BBL-PE (Biolegend) and CD360 (IL21R)-APC (Biolegend) for 30 minutes at room temperature. Subsequently, the cells were washed three times with PBS, and flow cytometry was used to confirm the expression of 4-1BBL and mbIL-21 on the surface of K562 cells.

[0192] K562 cells stably overexpressing 4-1BBL and mbIL-21 (purchased from ATCC) were sorted using a flow cytometry cell sorter. The simplified procedure is as follows: First, the transduced K562 cells were co-incubated with the flow cytometry antibodies Anti-4-1BBL-PE and CD360(IL21R)-APC at room temperature for 30 minutes. Then, the cells were washed twice with PBS and resuspended in PBS. Next, 4-1BBL+ and mbIL-21+ cells were sorted using a flow cytometry cell sorter (manufacturer: BD). Single cells were seeded into individual wells of a 96-well plate, with 200 μL of K562 complete culture medium (IMDM medium + 10% FBS) added to each well. After the single clones expanded to a certain number, they were transferred to T-25 culture flasks for further expansion. Simultaneously, 200 μL of cell suspension was used for flow cytometry to confirm the expression of 4-1BBL and mbIL-21 on their cell surface. The specific steps were as follows: K562 monoclonal cells were centrifuged, the supernatant was removed, and the cells were resuspended in PBS. Then, flow cytometry antibodies Anti-4-1BBL-PE and CD360(IL21R)-APC were added and co-incubated with the cells for 30 minutes. Afterward, the cells were washed three times with PBS and resuspended in PBS. Finally, the expression levels of 4-1BBL and mbIL-21 on the cell surface were detected using flow cytometry, and monoclonal cells with high and uniform expression of 4-1BBL and mbIL-21 were selected.

[0193] The activation function of different K562 monoclonal cells on NK cells was then examined. The specific steps were as follows: Expanded K562 feeder cells were harvested into centrifuge bottles, centrifuged to remove the supernatant, and resuspended in fresh complete culture medium to a density of 1E6–4E7 / mL. Cells were then transferred to an irradiator (manufacturer: Rad Source Technologies, Inc.) for irradiation at a dose of 100–300 Gy. Afterward, the cells were washed twice with PBS or physiological saline, resuspended in cryopreservation solution, and counted. The cell density was adjusted according to cryopreservation specifications (5E6 / mL–4E7 / mL), and the cells were cryopreserved in a programmed freezing system, then transferred to a gas-phase liquid nitrogen tank for later storage. Next, different K562 monoclonal cells were thawed and co-incubated with apheresis cells to activate the NK cells. Changes in NK cell purity and NK cell expansion folds were recorded from Day 0 to Day 14, and K562 monoclonal cells with high NK cell expansion folds and consistent performance across different donors were selected.

[0194] Monoclonal cells highly expressing 4-1BBL and mbIL-21 and effectively activating and promoting NK cell proliferation were selected and expanded to construct seed cell and master cell banks. Before production, one genetically modified K562 feeder cell was resuscitated from the master cell bank, expanded to a sufficient number, concentrated by centrifugation, and resuspended in K562 complete medium (IMDM medium + 10% FBS). The cells were then irradiated at a dose of 100–300 Gy. After irradiation, the cells were washed twice with PBS or physiological saline, resuspended in cryopreservation solution, and counted. The cell density was adjusted according to cryopreservation specifications (5E6 / mL–4E7 / mL), and the cells were cryopreserved in a programmed freezing system, then transferred to a gas-phase liquid nitrogen tank for later storage.

[0195] 1.2 Preparation process of gene-modified primary T cells

[0196] Fresh apheresis blood is prepared, aliquoted, and cryopreserved. On day 0, the cryopreserved blood is thawed in a 37°C water bath. After thaw, the blood is washed twice with physiological saline or PBS and then labeled and sorted using magnetic beads (Pan T isolation kit (Miltenyi biotec) or CD4 / CD8 beads (Miltenyi biotec)) to obtain primary T cells. On day 0, the T cells are co-incubated with an activation reagent (TransAct (Miltenyi biotec) or activation beads (Miltenyi biotec)). On day 2 or day 3, retroviruses containing the target gene (such as 4-1BBL and mbIL-21) are added for transduction. The transduced T cells are then transferred to culture flasks, G-rex containers, or culture bags for expansion culture. Once the cell number reaches the expected level (generally cultured to day 9-12), the transduced T cells are harvested, and the expression efficiency of the target molecule on their surface is detected. Simultaneously, the transduced primary T cells were harvested, and the cell suspension was concentrated into a transfer bag. After sealing with a heat sealer, the bag was placed in an irradiator for lethal dose irradiation. After irradiation, the cells were centrifuged to remove the supernatant, washed three times with physiological saline or PBS, resuspended in cryopreservation buffer, aliquoted into cryovials / bags, and transferred to a temperature-programmed freezer for cryopreservation. After cryopreservation, the cells were transferred to a gas phase liquid nitrogen tank for storage.

[0197] Example 2: Expansion of NK / CAR-NK cells

[0198] The frozen apheresis blood preparation and the K562 feeder cells from Example 1.1 were revived. The revived apheresis blood preparation and feeder cell preparation were mixed at a ratio of 1:1 (1:0.1 to 1:8 ratios can activate and expand NK cells), and seeded into culture flasks (G-Rex or culture bags can also be used). The mixture was cultured at 37°C in a 5% CO2 incubator, and this was recorded as Day 0. Depending on cell growth, complete NK cell culture medium (KBM581 medium (Corning) + 5% human AB serum (Akron Biotechnology) + 1000 IU / mL IL-2 (Jiangsu Jinshili Pharmaceutical Co., Ltd.) was added as needed. NK cell transduction was performed from Day 2 to Day 6. CAR was transduced into NK cells using lentivirus or retrovirus. A control group of cells was left untreated as an un-NK control. The BCMA CAR used in the examples of this disclosure is the BCMA CAR described in PCT patent application PCT / CN2024 / 072753. The CD19 / BCMA CAR used in the embodiments of this disclosure is the CD19 / BCMA CAR described in PCT patent application PCT / CN2024 / 144259. After transduction, un-NK and CAR-NK cells were transferred into culture flasks (G-Rex, culture bags, or bioreactors can also be used) for expansion culture, and culture medium and cytokines (such as IL-2, IL-15, etc.) were added according to the cell expansion. When the cells expanded to Day 6-21, un-NK / CAR-NK cells could be harvested. The harvested cells were centrifuged and the supernatant was discarded, or washed with physiological saline or PBS using automated washing and concentration equipment (such as Sepax C-pro or Sefia), resuspended in cryopreservation solution, and the cells were counted. After adjusting the density of CAR-NK or un-NK to 5E6-6E7 / mL, the cells were aliquoted into cryovials or cryopreservation bags and transferred to a programmed freezing system for cryopreservation. After cryopreservation, the cells were transferred to a gas phase liquid nitrogen tank for storage. Figure 1A shows the fold increase of CAR-NK cells harvested from donor 570 at different time points when cultured with K562 feeder cells transduced with 4-1BBL and mbIL-21. Figure 1B shows the fold increase of CAR-NK cells harvested from donor 571 at different time points when cultured with K562 feeder cells transduced with 4-1BBL and mbIL-21. The fold increase was calculated as follows: Fold increase = Total number of D14 cells * NK ratio / (Total number of D0 cells * NK ratio). As shown in Figures 1A and 1B, the fold increase of CAR-NK cells increased with increasing culture time.

[0199] Example 3: In vitro cytotoxic activity assay of NK / CAR-NK cells (feeder cells were modified K562).

[0200] Log-phase NK / CAR-NK cells obtained in Example 2 were collected into centrifuge tubes, centrifuged, and the supernatant was discarded. The cells were then washed twice with DPBS, resuspended in culture medium, and counted. Simultaneously, log-phase NCI-H929 cells (purchased from ATCC) were harvested, centrifuged, and the supernatant was discarded. The cells were washed once with PBS, stained with the CellTrace VIOLET proliferation kit (Invitrogen), resuspended in culture medium, and counted. CAR-NK cells and BCMA-expressing NCI-H929 tumor cells were seeded at effector-to-target ratios of 1:4, 1:16, and 1:64 into 96-well plates (for 4-hour killing) or 24-well plates (for repeated antigen stimulation killing assays, with an effector-to-target ratio of 1:4). A control group containing only CAR-NK cells was also included. After co-incubation for 20-24 hours, the residual proportion of tumor cells was determined by detecting tumor cell surface antigens or Celltrace, thereby calculating the percentage of tumor cell killing by NK / CAR-NK cells. The formula for calculating target cell lysis is: Kill percentage % = [1 - Celltrace BV421 + target cells / total cell] * 100%.

[0201] In addition, in the repeated antigen stimulation experiment, the supernatant of CAR-NK cells co-cultured with target cells NCI-H929 for 24 hours and 72 hours, as well as the supernatant of CAR-NK cells cultured alone, were harvested and frozen at -80°C for later use (for the detection of cytokine release).

[0202] Figure 2A shows the tumor clearance rate of BCMA CAR-NK cells harvested from Donor 570 at different time points after three consecutive rounds of killing NCI-H929 cells. Figure 2B shows the tumor clearance rate of BCMA CAR-NK cells harvested from Donor 570 at different time points after seven consecutive rounds of killing NCI-H929 cells. Since CAR-NK cells harvested on Days 10-21 could no longer kill tumor cells after the third round, these groups were removed from subsequent rounds, and only the groups harvested on Days 6-9 were compared. Figures 2C and 2D show the tumor clearance rates of BCMA CAR-NK cells harvested from Donor 571 at different time points during the continuous killing of NCI-H929 cells in the third and seventh rounds, respectively. As shown in Figures 2A-D, the in vitro killing ability of CAR-NK cells harvested at different in vitro culture time points weakened with prolonged culture time.

[0203] Simultaneously, in the repeated antigen stimulation experiment, BCMA CAR-NK / unNK cells harvested from different in vitro culture time points after three consecutive rounds (72 h) of killing NCI-H929 cells were counted. A separate group of BCMA CAR-NK / unNK cells harvested from different in vitro culture time points were also cultured and counted after 72 hours. Figure 3A shows the fold increase in BCMA CAR-NK cells harvested from Donor 570 at different time points after three consecutive rounds of killing NCI-H929 cells. Figure 3B shows the fold increase in BCMA CAR-NK cells harvested from Donor 570 at different time points after 72 hours of separate culture. Figure 3C shows the fold increase in BCMA CAR-NK cells harvested from Donor 571 at different time points after three consecutive rounds of killing NCI-H929 cells. Figure 3D shows the fold increase in BCMA CAR-NK cells harvested from Donor 571 at different time points after 72 hours of separate culture. As shown in Figures 3A-D, consistent with the expansion capacity of CAR-NK / unNK cells cultured alone, the expansion capacity of CAR-NK cells harvested at different in vitro culture time points in repeated antigen stimulation experiments decreased with the extension of in vitro culture time.

[0204] Example 4: Detection of cytokine secretion levels in NK / CAR-NK cells (feeder cells were modified K562 cells).

[0205] Cell supernatants collected in the in vitro killing assay were removed from a -80°C freezer and thawed at 4°C. After complete thawing, the supernatants were diluted with culture medium according to experimental requirements. Specifically, the cell supernatants from the 24-hour killing assay were diluted 5-fold, and those from the 72-hour killing assay were diluted 10-fold for subsequent experiments. The kit used in this experiment was the INF-γHTRF detection kit (company: CISBIO). Following the kit instructions, the standard stored at -80°C was diluted 3-fold with 1× working solution to the initial detection concentration Std7. Subsequently, a serial dilution method (each dilution factor of 2-fold) was used to sequentially dilute the Std7 standard solution 6 times to obtain Std6, Std5, Std4, Std3, Std2, and Std1 standard solutions; Std0 was the 1× working solution. The standard solutions from Std7 to Std0 were added to 384-well white plates at a volume of 10 μL / well. Simultaneously, the diluted cell supernatant was added to the 384-well plate at a rate of 10 μL / well. Next, the antibody, stored at -80℃, was removed, mixed thoroughly, and then diluted 40-fold with the detection reagent. For example, 10 μL each of Eu Cryptate antibody and XL antibody were mixed thoroughly and added to 780 μL of the detection reagent. Then, the diluted antibody was added to the 384-well plate at a rate of 8 μL / well. After sample addition, the 384-well plate was centrifuged at 1000 rpm for 1 minute, sealed with a sealing film, and incubated overnight at 23℃. The next day, the 384-well plate was read using a multi-plate reader. Finally, the concentration of IFN-γ was calculated based on the standard curve (the standard curve fits a linear relationship y = aX + b, and R² ≥ 0.99).

[0206] As shown in Figures 4A-F, the cytokine secretion levels of CAR-NK cells harvested at different in vitro culture time points decreased with prolonged culture time. Figure 4A shows the INF-γ secretion levels of BCMA CAR-NK / unNK cells harvested from Donor 570 at different time points after 72 hours of stimulation with and without tumor cells. Figure 4B shows the INF-γ secretion levels of BCMA CAR-NK / unNK cells harvested from Donor 571 at different time points after 72 hours of stimulation with and without tumor cells.

[0207] Example 5: Detection of metabolic activity of NK / CAR-NK cells (feeder cells were modified K562 cells)

[0208] Energy metabolism is crucial for cellular function, playing a vital role in numerous key cellular processes. The inventors employed a seahorse assay to evaluate the energy metabolism activity of BCMA CAR-NK cells harvested at different culture time points. The kit used in this experiment was the Agilent Seahorse XF Glycolytic Stress Assay Kit. Specific procedures are detailed in the kit instructions; in short, CAR-NK cells harvested at different in vitro culture times were seeded in seahorse culture plates (Seahorse Bioscience) and cultured at 37°C in a non-CO2 incubator for 40 minutes. Cells were then treated with glucose, oligomycin, and 2-deoxy-D-glucose (2-DG) according to the kit instructions. The extracellular acidification rate (ECAR) was then measured using the XF96 Seahorse Bioscience extracellular flux analyzer.

[0209] As shown in Figures 5A-D, the metabolic activity of CAR-NK cells decreased with prolonged in vitro culture time, and both glycolytic capacity and potential also decreased with extended in vitro culture time. Figure 5A shows the basal metabolic activity of CAR-NK cells harvested from Donor 570 at different in vitro culture time points. Figure 5B shows the basal metabolic activity of CAR-NK cells harvested from Donor 571 at different in vitro culture time points. Figure 5C shows the glycolytic capacity of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points. Figure 5D shows the glycolytic potential of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points.

[0210] In addition, the metabolic capacity of cryopreserved CAR-NK cells harvested at different time points after thawing was examined. Thawed CAR-NK cells were seeded into 24-well plates at an effector cell:target cell (NCI-H929 cells) ratio of 1:1 and cultured overnight at 37°C in a 5% CO2 incubator. CAR-NK cells were then collected, and the same seahorse assay was performed to detect their extracellular acidification rate (ECAR). As shown in Figures 6A-6D, the metabolic activity of CAR-NK cells harvested at different in vitro culture time points decreased after antigen stimulation with increasing in vitro culture time. Furthermore, the glycolytic capacity and potential also decreased with prolonged in vitro culture time. Figure 6A shows the basal metabolic activity of CAR-NK cells harvested from Donor 570 at different in vitro culture time points after antigen stimulation. Figure 6B shows the basal metabolic activity of CAR-NK cells harvested from Donor 571 at different in vitro culture time points after antigen stimulation. Figure 6C shows the glycolytic capacity of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points after antigen stimulation. Figure 6D shows the glycolytic potential of CAR-NK cells harvested from Donor 570 and Donor 571 at different in vitro culture time points after antigen stimulation.

[0211] Example 6: Phenotypic differences in BCMA CAR-NK cells (feeder cells were modified K562) harvested at different in vitro culture times.

[0212] After thawing frozen BCMA CAR-NK cell preparations, cells were washed three times with PBS and resuspended in staining buffer (PBS + 0.5% FBS or BSA). After counting, cells were allocated to 96-well plates (1–5E5 cells per well) according to cell density. The corresponding flow cytometry antibodies (Anti-CD25-PE, Biolegend; Anti-CD16-PE, Biolegend; Anti-NKG2C-APC, MILTENYI BIOTEC; Anti-CD57-APC, Biolegend) were added, and the cells were incubated at room temperature in the dark for 30 minutes. Cells were centrifuged at 300g for 6 minutes, the supernatant was discarded, and the cells were washed twice with 200 μL of staining buffer. Finally, the cells were resuspended in 150 μL of staining buffer and analyzed by flow cytometry.

[0213] As shown in Figures 7A-7D, CAR-NK cells harvested at different in vitro culture time points exhibited different phenotypes. With prolonged in vitro culture time, the expression of CD16 and NKG2C gradually increased, while CD25 expression gradually decreased, showing a significant decrease by day 10 until it was completely absent. The NK cell maturation marker CD57 maintained low expression levels from day 6-10, then gradually increased with prolonged in vitro culture time. Figure 7A shows the expression level of CD16 in CAR-NK cells harvested at different in vitro culture time points. Figure 7B shows the expression level of CD25 in CAR-NK cells harvested at different in vitro culture time points. Figure 7C shows the expression level of CD57 in CAR-NK cells harvested at different in vitro culture time points. Figure 7D shows the expression level of NKG2C in CAR-NK cells harvested at different in vitro culture time points.

[0214] CD25 is a cell surface protein encoded by the human gene IL2RA, which, along with the β subunit (CD122) and γ subunit (CD132), forms the interleukin-2 receptor. Resting T cells, NK cells, B cells, and monocytes express very little CD25. However, when T cells or NK cells are activated, CD25 expression is rapidly upregulated. As shown in Figure 7A, CD25 expression in CAR-NK cells decreases slowly with prolonged in vitro culture time, then rapidly declines after day 10. This is similar to the changing trends in the metabolic and cytotoxic abilities of CAR-NK cells.

[0215] CD57 is expressed on T cells and NK cell subsets and is a molecule associated with cell differentiation. Some studies suggest that CD57 is a marker for NK cells with poor proliferative capacity and potential immunosenescence. As shown in Figure 7B, the CD57 level on the surface of CAR-NK cells increases rapidly after day 10 with prolonged in vitro culture time, indicating that CAR-NK cells are gradually entering the senescence process (or terminal differentiation state).

[0216] CD16 is an important biomarker expressed on the surface of NK cells and mediates ADCC. In immunology, NK cells can be divided into two classic subsets: CD56 and CD16. dim CD16 + NK cells and CD56 bright CD16 neg / dim NK cells. CD56 dim CD16 + NK cells account for approximately 90% of circulating NK cells and are considered a mature NK subset; CD56 bright CD16 neg / dimNK cells comprise approximately 10% and are considered an immature NK subset. As shown in Figure 7C, CAR-NK cells harvested on or before day 9 exhibit low CD16 expression, but upon entry into mice, CD16 expression rapidly upregulates and exhibits ADCC activity (data not shown).

[0217] Example 7: In vivo antitumor activity assay of CAR-NK cells (feeder cells were modified K562).

[0218] To evaluate the persistence and antitumor activity of CD19 / BCMA CAR-transduced NK cells in vivo, we used a NOD / SCID IL-2Rγnull (NSG) xenograft model and the invasive Nalm-6 cell line (purchased from ATCC). Mouse experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee.

[0219] NSG mice (10-12 weeks old; Jiangsu Jicui Pharmaceutical Co., Ltd.) were irradiated with 300 cGy on day 0 and fed with FFLuc-labeled Nalm-6 cells (2 × 10⁻⁶). 5 Intravenous (iv) inoculation. Seven days post-inoculation, expanded CAR-NK cells, UN-NK cells, or HBSS were injected via tail vein. Mice underwent bioluminescence imaging (BLI: IVIS Lumina LT imaging system, PerkinElmer) weekly. The photon flux rate within the normalized region of interest was determined using software, and signal quantification was performed in photons per second (p / s). As shown in Figure 8, the tumor clearance capacity of CAR-NK cells harvested at different in vitro culture time points in the mouse model decreased with prolonged culture time. Specifically, CAR-NK cells harvested on day 6 in the 2M dose group resulted in toxicity and death in mice between days 10 and 17 due to excessive dosage. However, in the corresponding low-dose group (0.5M), mice not only maintained efficacy but also exhibited better safety. This suggests that for CAR-NK cells harvested at earlier culture time points, the dosage can be reduced to achieve better safety.

[0220] To assess the pharmacokinetic (PK) potential of CD19 / BCMA CAR-NK cell products harvested at different in vitro culture times in a mouse model, the following experiment was conducted: On days 4, 10, and 17 after CAR-NK cell infusion, 200 μL of peripheral circulating blood was collected from mice. Half of the blood was used for complete blood count (CBC) to obtain the absolute number of white blood cells. The other half was used for flow cytometry. Anti-human CD45, Anti-human CD3, Anti-human CD56, and Anti-ScFv-APC antibodies were added, and the cells were incubated at room temperature for 30 minutes. Red blood cells were then lysed, washed twice, and the CD45 content in white blood cells was detected using flow cytometry. + CD3 - CD56 + ScFv + The proportion of CAR-NK cells was calculated. Multiplying this proportion by the number of WBCs yields the absolute number of CAR-NK cells in the mouse at that time point. As shown in Figure 9, the amplification capacity of CD19 / BCMA CAR-NK cell products harvested at different in vitro culture time points decreased in the mouse model with increasing culture time. Specifically, CAR-NK cells harvested on day 6 in the 2M dose group caused toxicity and death in mice between days 10 and 17 due to the excessively high dose. However, in the corresponding low-dose group (0.5M), the amplification of CAR-NK cells in mice reached a level similar to or even higher than that of the 2M group on day 10, and the amplification capacity and efficacy showed a consistent trend.

[0221] Example 8: In vitro killing activity assay of CAR-NK cells (feeder cells were modified primary T cells)

[0222] Preparation of CAR-NK cells (feeder cells are modified primary T cells): The frozen apheresis blood preparation and the modified primary T cells from Example 1.2 were thawed and mixed at a 1:1 ratio (a ratio of 1:0.1–1:8 can activate and expand NK cells). The mixture was then seeded into culture flasks (G-Rex or culture bags could also be used) and cultured at 37°C in a 5% CO2 incubator, recorded as Day 0. Depending on cell growth, complete NK cell culture medium (KBM581 medium (Corning) + 5% human AB serum (Akron Biotechnology) + 1000 IU / mL IL-2 (Jiangsu Jinsili Pharmaceutical Co., Ltd.) was added as needed. From Day 2 to Day 6, NK cells were sorted and transduced. CAR cells were transduced into NK cells using lentivirus or retrovirus. One group of cells was left untreated as an un-NK control. The BCMA CAR used in the embodiments of this disclosure is the BCMA CAR described in PCT patent application PCT / CN2024 / 072753. After transduction, un-NK and CAR-NK cells were transferred into culture flasks (G-Rex, culture bags, or bioreactors can also be used) for expansion culture, and culture medium and cytokines (such as IL-2, IL-15, etc.) were added according to the cell expansion. When the cells expanded to Day 6-21, un-NK / CAR-NK cells could be harvested. The harvested cells were centrifuged and the supernatant was discarded, or washed with physiological saline or PBS using automated washing and concentration equipment (such as Sepax C-pro or Sefia), resuspended in cryopreservation solution, and the cells were counted. After adjusting the density of CAR-NK or un-NK to 5E6-6E7 / mL, the cells were aliquoted into cryovials or cryopreservation bags and transferred to a programmed freezing system for cryopreservation. After cryopreservation, the cells were transferred to a gas phase liquid nitrogen tank for storage.

[0223] In vitro killing assay procedure: After unpacking and thawing, the frozen NK / CAR-NK cells from the above steps were collected into centrifuge tubes. After centrifugation and discarding the supernatant, the cells were washed twice with DPBS, resuspended in culture medium, and counted. Simultaneously, NCI-H929 cells in the logarithmic growth phase were harvested, centrifuged, and the supernatant was discarded. After washing once with DPBS, the target cells were stained with a CELLTRACE VIOLET proliferation kit (Invitrogen), resuspended in culture medium, and counted. CAR-NK cells and NCI-H929 tumor cells expressing BCMA antigen were seeded at an effector-to-target ratio of 1:4 in 24-well plates for repeated antigen stimulation killing assays. A control group containing only CAR-NK cells was also set up. After co-incubation for 20-24 hours, the residual proportion of tumor cells was determined by detecting tumor cell surface antigen or CELLTRACE, thereby calculating the percentage of tumor cell killing by NK / CAR-NK cells. The formula for calculating target cell lysis is: Kill percentage % = [1 - Celltrace BV421 + target cells / total cell] * 100%.

[0224] Figure 10 shows the ability of BCMA CAR-NK cells harvested at different time points from genetically modified primary T cells as feeder cells to kill NCI-H929 cells in vitro. As can be seen from the in vitro repeated antigen stimulation experiment, consistent with NK cells cultured using K562 as feeder cells, the in vitro killing ability of CAR-NK cells harvested at different in vitro culture time points from genetically modified primary T cells as feeder cells decreased with increasing in vitro culture time.

[0225] Example 9: In vivo antitumor activity assay of CAR-NK cells (feeder cells were modified primary T cells)

[0226] To evaluate the in vivo persistence and antitumor activity of BCMA CAR cells prepared using a modified primary T cell process, we employed a NOD / SCID IL-2Rγnull (NSG) xenograft model and an invasive NCI-H929 cell line. Mouse experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee. NSG mice (10–12 weeks old; Jiangsu Jicui Pharmaceutical Co., Ltd.) were irradiated with 300 cGy on day 0 and fed with FFLuc-labeled NCI-H929 cells (2 × 10⁻⁶ cells). 5Intravenous (iv) inoculation. Fourteen days post-inoculation, expanded CAR-NK cells, UN-NK cells, or HBSS were injected via tail vein. Mice underwent bioluminescence imaging (BLI: IVIS Lumina LT imaging system, PerkinElmer) weekly. Photon flux rate within the normalized region of interest was determined using software, and signal quantification was performed in photons per second (p / s). In selected experiments, mice injected with UN-NK cells were simultaneously injected intraperitoneally with a low dose of IL-15 (Peprotech, 0.5 μg / mouse), followed by injections every 2–3 days for two weeks as per the established protocol. As shown in Figure 11, the tumor clearance capacity of CAR-NK cells harvested at different in vitro culture time points in the mouse model decreased with prolonged culture time.

Claims

1. A method for preparing natural killer (NK) cells, the method comprising co-culturing modified feeder cells with NK cell seed cells, wherein the NK cells are cultured for no more than 10 days.

2. The method of claim 1, wherein, The NK cells were cultured for 4 to 10 days. Preferably, the NK cells are cultured for 6 to 9 days; More preferably, the NK cells are cultured for 6, 7, 8 or 9 days.

3. The method of claim 1 or 2, wherein, The seed cells are selected from peripheral blood samples, peripheral blood mononuclear cell (PBMC) samples, umbilical cord blood samples, umbilical cord blood mononuclear cell (CBMC) samples, lymphocyte samples, leukocyte samples, apheresis products, leukocyte apheresis products, whole blood samples, erythrocyte sedimentation rate (ESR) brown-yellow layer samples, enriched or isolated primary NK cells, or NK cell lines. Preferably, the seed cells are selected from apheresis products, leukocyte apheresis products, cord blood samples, cord blood mononuclear cell (CBMC) samples, peripheral blood samples, or peripheral blood mononuclear cell (PBMC) samples.

4. The method according to any one of claims 1-3, wherein, The feeder cells are cell lines or primary cells.

5. The method according to any one of claims 1-4, wherein, The feeder cells are K562, Jurkat, HuT-78, 721.221, SupT-1, or Nalm-6.

6. The method according to any one of claims 1-4, wherein, The feeder cells are primary T cells.

7. The method according to any one of claims 1-6, wherein, The modified feeder cells were inactivated by irradiation.

8. The method according to any one of claims 1-7, wherein, The feeder cells express at least one of the following: 4-1BB binding protein (such as 4-1BB ligand (4-1BBL) or 4-1BB antibody), membrane-bound interleukin-21 (mbIL-21), CD226 binding protein (such as CD226 antibody), NKG2A binding protein (such as NKG2A antibody), NKG2D binding protein (such as NKG2D antibody), IL-15 or its fusion protein with IL-15 receptor α (IL-15Rα), NKp30 binding protein (such as NKp30 antibody), IL-7, and IL-2.

9. The method according to any one of claims 1-8, wherein, The feeder cells expressed 4-1BB ligand (4-1BBL) and membrane-bound interleukin-21 (mbIL-21).

10. The method according to any one of claims 1-9, wherein, The feeder cells expressed: 4-1BBL and mbIL-21; CD226 antibody, 4-1BBL and mbIL-21; NKG2A antibody, 4-1BBL and mbIL-21; NKG2D antibody, 4-1BBL and mbIL-21; IL-15 and IL-15Rα fusion protein, 4-1BBL and mbIL-21; 4-1BB antibody and mbIL-21; NKp30 antibody, 4-1BBL and mbIL-21; IL-7; IL-2 and IL-7; 4-1BBL and IL-2; 4-1BBL; 4-1BBL and IL-7; IL-15; 4-1BBL and IL-15; or, IL-2 and IL-15.

11. The method according to any one of claims 1-10, wherein, The seed cells and the modified feeder cells are cultured together at a ratio of 1:0.1 to 1:

100.

12. The method of claim 11, wherein, The seed cells are enriched or isolated primary NK cells.

13. The method according to any one of claims 1-12, wherein, The seed cells and the modified feeder cells were cultured together at a ratio of 1:0.1 to 1:

10. Preferably, the ratio of the seed cells to the modified feeder cells is 1:0.5 to 1:4; More preferably, the ratio of the seed cells to the modified feeder cells is 1:0.5 to 1:2, for example 1:0.5 to 1:1, 1:0.5 to 1:1.5, 1:1 to 1:2, 1:1.5 to 1:2, for example 1:

1.

14. The method of claim 13, wherein, The seed cells are peripheral blood mononuclear cell (PBMC) samples or products of apheresis.

15. The method according to any one of claims 1-14, wherein, The NK cells were prepared in a culture medium supplemented with cytokines; Preferably, the cytokine is selected from one or more of IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, and IL-21.

16. The method according to any one of claims 1-15, wherein, The method also includes introducing heterologous nucleic acids into NK cells or their seed cells.

17. The method of claim 16, wherein, The heterologous nucleic acid encodes a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

18. The method of claim 17, wherein, The chimeric antigen receptor (CAR) specifically binds to the target antigen.

19. The method of claim 18, wherein, The target antigens are selected from BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folic acid receptor-α, GD2, G D3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 fine adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, Claudin 18.2, Claudin 6, NKG2D, DLL3, CD70, CS-1, c-Met, glycolipid FF77, PD-L1 and PD-L2.

20. The method according to any one of claims 16-19, wherein, The heterologous nucleic acid is delivered via a lentiviral vector or a retroviral vector.

21. Natural killer (NK) cells, prepared by the method of any one of claims 1-20.

22. The NK cell of claim 21, wherein, The NK cells had a higher CD25 positivity rate compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days.

23. The NK cell of claim 21 or 22, wherein, The NK cells had a lower CD57 positivity rate compared to NK cells prepared by co-culturing with the same feeder cells for more than 16 days.

24. The NK cells according to any one of claims 21-23, wherein, The NK cells had a lower CD16 positivity rate compared to NK cells prepared by co-culturing with the same feeder cells for more than 12 days.

25. The NK cells according to any one of claims 21-24, wherein, The NK cells secreted higher levels of IFN-γ compared to NK cells cultured for more than 12 days using the same feeder cells.

26. The NK cells according to any one of claims 21-25, wherein, The NK cells exhibited stronger cytotoxicity compared to NK cells cultured for more than 12 days using the same feeder cells.

27. The NK cells according to any one of claims 21-26, wherein, Compared to NK cells cultured for more than 12 days using the same feeder cells, the NK cells exhibited a stronger ability to expand after being administered to the subject.

28. The NK cells according to any one of claims 21-27, wherein, Compared to NK cells cultured for more than 12 days using the same feeder cells, the NK cells exhibited stronger glycolytic capacity and / or glycolytic potential.

29. The NK cells according to any one of claims 21-28, wherein, At least a portion of the NK cells were further engineered to express IL-15.

30. The NK cells according to any one of claims 21-29, wherein, The IL-15 is either secreted IL-15 or membrane-bound IL-15 (membrane-bond IL-15, mbIL-15).

31. An NK cell suitable for administration to a subject in need, wherein... (1) At least 40% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%) of NK cells express CD25; (2) Up to 50% (e.g., up to 40%, up to 30%, up to 20%, up to 10%, or 0%) of NK cells express CD57; and / or (3) Up to 80% (e.g., up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, up to 10% or 0%) of NK cells express CD16.

32. A pharmaceutical composition comprising NK cells as described in any one of claims 21-30 or NK cells as described in claim 31, and optionally a pharmaceutically acceptable carrier and / or excipient.

33. A method for treating a disease or condition, the method comprising administering to an individual in need an effective amount of NK cells according to any one of claims 21-30, NK cells according to claim 31, or a pharmaceutical composition according to claim 32; Preferably, the disease or symptom is a tumor or an autoimmune disease.

34. The method of claim 33, wherein, The cells used are either autologous or allogeneic.

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